A single-nucleotide substitution of CjTKPR1 determines pollen production in the gymnosperm plant Cryptomeria japonica

Abstract Pollinosis, also known as pollen allergy or hay fever, is a global problem caused by pollen produced by various plant species. The wind-pollinated Japanese cedar (Cryptomeria japonica) is the largest contributor to severe pollinosis in Japan, where increasing proportions of people have been affected in recent decades. The MALE STERILITY 4 (MS4) locus of Japanese cedar controls pollen production, and its homozygous mutants (ms4/ms4) show abnormal pollen development after the tetrad stage and produce no mature pollen. In this study, we narrowed down the MS4 locus by fine mapping in Japanese cedar and found TETRAKETIDE α-PYRONE REDUCTASE 1 (TKPR1) gene in this region. Transformation experiments using Arabidopsis thaliana showed that single-nucleotide substitution (“T” to “C” at 244-nt position) of CjTKPR1 determines pollen production. Broad conservation of TKPR1 beyond plant division could lead to the creation of pollen-free plants not only for Japanese cedar but also for broader plant species.


Introduction
Pollen production is critical for plant reproduction; therefore, the genetics of pollen development have been studied extensively. This research has led to the discovery of the pollen developmental pathway, as well as many genes involved in pollen development regulation (1). Several gene mutants give rise to a pollen-free phenotype called male sterility (1,2). In agriculture, pollen production is also important. Many pollen grains are required for artificial pollination and efficient seed/fruit production. Conversely, male sterility is a crucial trait required to produce hybrid crops with stable quality and quantity (3). For our health, pollen can sometimes be harmful by triggering an allergic reaction, pollinosis (4,5).
Japanese cedar is a wind-pollinated conifer and is the most important timber species in Japan, accounting for 58% (12.7 million m 3 ) of total roundwood production in 2019 (16). However, Japanese cedar is also the leading cause of the most severe allergic reactions to pollen in Japan (11). A single Japanese cedar tree typically produces thousands of male strobili, which correspond to male flowers in angiosperms; a single male strobilus contains approximately 100,000-300,000 pollen grains ( Fig. 1A-C; Movie S1) (17). Upon release, these vast numbers of pollen grains are carried away by the wind. The proportion of people suffering from pollinosis caused by Japanese cedar pollen in Japan has increased in recent years, from 16.2% in 1998 to 26.5% in 2008 and 38.8% in 2019 (18,19). Therefore, planting pollen-free trees is a promising strategy for maintaining Japanese cedar as a building material while countering its effects in pollinosis. According to these situations and demand, several pollen-free cultivars were bred (20), and the annual production of seedlings of low-pollen and pollen-free Japanese cedar has been increasing yearly in Japan (21). Unveiling the genetic mechanism of pollen-free mutants will contribute not only to promoting pollen-free timber breeding but also to understanding the flowering mechanism in gymnosperm plants.
Conifers have large genomes, which complicates genetic studies of these species (e.g. 25.4 Gb for Pinus tabuliformis (22) and ∼20 Gb for Picea abies (23)). Flowering trait analyses are particularly problematic due to the necessity of large fields for keeping materials for segregation analyses, as well as a wait time of several years until the flowering stage can be examined (24). Among conifers, Japanese cedar has several advantages for flowering trait analyses. Its genome is smaller (∼10.8 Gb) (25) than those of other conifer species, and its flowering can be induced artificially using gibberellic acid (26). Previous field studies identified four male sterility loci, i.e. MALE STERILITY 1-4 (MS1-MS4), in Japanese cedar (27,28), through linkage analyses (28,29). Recently, a strong candidate gene for MS1 was discovered by our research (30). Two different mutation alleles of CJt020762, which are located on the MS1 locus, showed pollen-free phenotype, suggesting that CJt020762 is a causal gene of MS1, although functional validation is required. Here, we focus on identifying the causal gene in the MS4 locus. We find abnormal pollen wall structure at the microspore stage in the ms4 mutant. Fine-mapping and RNA-sequencing (RNA-seq) analysis suggest the CjTKPR1 gene is the strong candidate gene for the causal gene of MS4. Furthermore, functional validation with Arabidopsis thaliana reveals single-nucleotide substitution of CjTKPR1 determines pollen production.

Male strobilus and pollen observation of ms4 mutant
The ms4 mutant "Shindai-8" was discovered in a planted forest in Niigata Prefecture, Japan. Mature male strobili of homozygous ms4 mutants (ms4/ms4) produced almost no pollen (Fig. 1A-G). Very little of the pollen produced by the ms4 mutants showed obvious abnormal phenotypes compared to wild-type (WT) plants, such as small or collapsed pollen grains ( Fig. 1H and I). Scanning electron microscope (SEM) observations revealed that the ms4 mutant produces small, abnormal orbicules, which are components of pollen wall (Fig. 1J-M) (31). Furthermore, transmission electron microscope (TEM) observation a showed thick ectexine structure of pollen wall in WT as previously reported ("Ec" in Fig. 1N (32)). On the other hand, ms4 mutant pollen had degenerated ectexine ("dEc" in Fig. 1O). Pollen wall is an essential constituent of the pollen, and dysfunctional biosynthesis genes often result in male sterility (33,34).

Fine mapping of MS4 locus and transcriptome analysis
To narrow down the region of MS4 locus, we generated a segregating population by crossing the ms4 mutant with heterozygous individuals ("Sindai-8" × "S8HK5"; Table S1). We found that 46 individuals produced normal pollen, whereas 48 showed pollenfree phenotype (Table S2). Then, we conducted fine mapping using these 94 individuals and narrowed down MS4 for an approximately 7.65-Mb-wide region on linkage group 4 ( Fig. 2A). Annotation analysis revealed 67 genes in this region ( Fig. 2B and Table S3). Among these genes, we found a homologous gene of TETRAKETIDE α-PYRONE REDUCTASE 1 (TKPR1) (hereafter CjTKPR1); this gene is essential for sporopollenin biosynthesis through making the exine layer of the pollen wall (33,36). RNA-seq data revealed that CjTKPR1 had the highest expression levels in all three male strobilus samples (183.17-1,131.88 transcripts per million [TPM]), but less or no expression in inner bark and leaf tissues (0 and 0.54 TPM, respectively; Fig. 2B and Table S3) (35).

Male organ-specific expression and sequence comparison of TKPR1 suggest that TKPR1 is causal gene of MS4
TKPR1 is expressed in the tapetum, and tetraketide reductase activity of TKPR1 was observed in vitro for A. thaliana and tobacco (37,38). Mutants of TKPR1 lead to male sterility in several angiosperm species such as A. thaliana, rice, tobacco, and gerbera (36)(37)(38)(39). Histological observations have revealed that mutants of AtTKPR1 and OsTKPR1 show no obvious defects at the tetrad stage, although abnormal microspores appeared in later stages and pollen cells, before degenerating rapidly (39,40). CjTKPR1 shares 54.6-64.9% amino acid identity with A. thaliana, gerbera, tobacco, and rice (Figs. 3A and B and S1). Reverse-transcription polymerase chain reaction (RT-PCR) showed specific CjTKPR1 expression in the male strobilus from WT and ms4 mutant (Fig. 3C). Furthermore, in situ hybridization of male strobili confirmed CjTKPR1 expression in the tapetum of Japanese cedar ( Fig. 3D-G). Histological observations of male strobili showed that the ms4 mutant not only has a clear tetrad structure but also has crushed, abnormal microspores ( Fig. 3H-M) as previously observed in A. thaliana and rice (39,40). These results support the hypothesis that CjTKPR1 is the causal gene of MS4 in Japanese cedar.
Sequence comparison of CjTKPR1 between WT ("Higashikanbara-5") and ms4 ("Shindai-8") revealed only four nucleic acid substitutions; interestingly, only one amino acid substitution was detected at position 244 nt (C82R; Fig. 3A and B) among these substitutions. This single-nucleotide polymorphism (SNP) at 244 nt was completely linked with the pollen-free phenotype in wild accessions (Table S1) and two crossing progenies ("S8" × "S8HK5," Table S2; selfing progenies of "S8DY1," Table S4), except for mutants from other male sterility loci (ms1-ms3). Thus, only plants with C/C at position 244 nt showed the pollenfree phenotype, and those having T/T or T/C showed normal pollen production (Tables S1, S2, and S4). This cysteine-82 of CjTKPR1 was perfectly conserved in various plant species, including both angiosperms and gymnosperms (Figs. 3B and S1). Furthermore, evaluation tools for amino acid substitution predicted that C82R leads to severe defects for CjTKPR1 ( Fig. S2 (42, 43)). These results suggest that CjTKPR1 is an important gene for pollen production in Japanese cedar and that the nucleotide substitution at position 244 nt (C82R) is a strong candidate for the causal mutation of MS4 in Japanese cedar.

Functional analysis of CjTKPR1 using A. thaliana
We conducted complementation tests to confirm that CjTKPR1 sequences control pollen production using A. thaliana (Fig. 4). First, we obtained the TKPR1 mutant of A. thaliana (Attkpr1-1 mutant, SAIL_837_D01), which homozygous plant shows a pollen-free phenotype ( Fig. 4B and C) (37). Then, four CjTKPR1 sequences, i.e. the WT, ms4 mutant (MT), WT background with a point mutation at 244 nt (T to C, WM), and ms4 background with a point mutation at 244 nt (C to T, MW) (Fig. 4A), were each introduced into heterozygous plants of AtTKPR1 (AtTKPR1/Attkpr1-1), not into Attkpr1-1 homozygous plants (Attkpr1-1/Attkpr1-1) that produce no pollen and therefore obtain no progenies. Next, we selected Attkpr1-1 homozygous plants with CjTKPR1 constructs from the T2 or T3 generations. Anthers of these plants were stained by Alexander staining and pollen production was confirmed (44). As a result, CjTKPR1-WT-introduced plants recovered pollen production (Fig. 4D), whereas CjTKPR1-MT-introduced plants still showed the pollen-free phenotype (Fig. 4E). These results indicate that CjTKPR1-WT can complement pollen production in A. thaliana and that the CjTKPR1 allelic difference controls pollen production. Lines with a single-nucleotide substitution at 244 nt showed that CjTKPR1-WM-introduced plants, which have a CjTKPR1-WT background with a point mutation at 244 nt, could not recover pollen production (Fig. 4F). However, CjTKPR1-MW-introduced plants, which have a ms4 background without a point mutation at 244 nt, recovered pollen production (Fig. 4G). Together, these complementation test results indicate that single-nucleotide substitution of CjTKPR1 at 244 nt determines pollen production.

Discussion
In this study, we identified a single-nucleotide substitution of CjTKPR1 as a causal mutation of MS4 in Japanese cedar. This knowledge provides the basic information for selecting and breeding pollen-free Japanese cedar efficiently and precisely. Our results also contribute for the creation of new pollen-free plants using genome editing by targeting TKPR1 gene. Cupressaceae species are suitable target plant species because they are often used for timber but are also global sources of severe pollinosis including Arizona cypress, Italian cypress, and mountain cedar (15). Since the recent progress of genome-editing methods that enable the application of mutagenesis for broader plant species, including Japanese cedar (45), combining our results with genome editing methods will accelerate the creation of new pollen-free plants.
Tapetum expression of CjTKPR1 in both WT and ms4 mutant plants suggests that CjTKPR1 of ms4 may be dysfunctional at the protein level, but not at the transcription level, for example in terms of tetraketide reductase activity (Fig. 3D-G). This study identified the cause of dysfunction of TKPR1 activity at the single amino acid level. Because the activity domain of TKPR1 has not yet been analyzed well, our results give a new insight into the enzymatically important amino residues for tetraketide reductase activity of TKPR1.
Interestingly, TKPR1 occurs not only in seed plants but also in phylogenetically basal plants without pollen, such as the fern Ceratopteris richardii and liverwort Marchantia polymorpha (Fig. S1). Although ferns and liverworts produce sperm as male gametes and do not have a pollen wall structure, they have a perispore layer within the spore that consists of sporopollenin. Sporopollenin is thought to be a key structure for the colonization of terrestrial environments during plant evolution, protecting them from ultraviolet B light (46,47). Our results that CjTKPR1 could rescue dysfunction of TKPR1 of A. thaliana may imply that other pollen developmental genes are also conserved among the Plantae. Further investigation of the pollen developmental genes of Japanese cedar and comparison thereof among other plant species will help elucidate the evolution of male gametes in plants.

Plant materials
Male strobili of Japanese cedar were collected from planted fields. We used 10 lines of wild accessions, three crossing parents (Table S1), and two combinations of crossing progenies (Tables  S2 and S4). Detailed information including sample name, sampling location, pollen phenotype, and SNP types at 244 nt of CjTKPR1 for all the Japanese cedar materials used in this study is listed in Tables S1, S2, and S4. DNA and RNA were extracted from needle (29) and male strobilus (30) tissues, as described previously.

Male strobilus and pollen observations
For the male strobilus observations, WT ("Higashikanbara-5") and ms4 mutant ("Shindai-8") of Japanese cedar plants were used. Male strobili were cut using a razor and photographed under a stereomicroscope (SZ-11; Olympus, Tokyo, Japan) using a digital camera (Wraycam EL310; Wraymer, Osaka, Japan). Pollen grains were stained with Alexander staining solution (38) and photographed under a light microscope (BX-50; Olympus). Pollen grains from "Higashikanbara-5" and "Shindai-8" were counted using a cell counter (48). Briefly, a single male strobilus was gently squashed using a pestle and suspended with water. The pollen suspension was then filtrated through two mesh filters (100 and 20 μm) to remove male strobilus debris and dust. The cleaned suspension was mixed with a cell counter buffer (CASYton; OLS, A B Fig. 2. Fine mapping and analysis of candidate genes in the MS4 locus. A) Fine mapping of the MS4 locus according to a linkage map around MS4 (top) and physical map of the MS4 region (bottom). B) Expression patterns of 67 genes of the MS4 region from male strobili (three samples) or inner bark and leaf tissues (two samples). RNA-seq data were obtained from our previous study (35) and expression levels were categorized into six levels (0, >0-1, >1-10, >10-100, >100-1,000, or >1,000 TPM). Gene no. 52, CjTKPR1, had the highest expression level among male strobilus samples but no or little expression was detected in inner bark or leaf tissues. Precise expression data for each gene are provided in Table S3.
Bremen, Germany), and pollen grains were counted using a CASY cell counter (OLS).

SEM
Pollen grains were extracted from progenies of "S8" × "S8HK5" ("P411" as normal pollen and "P380" as ms4 mutant; Table S1). Male strobili were harvested on 2019 December 12, after pollen maturation but before pollen release. After washing with deionized water, the male strobili were air-dried for 2 days. Pollen was harvested from dried male strobili and further sifted through a 75-μm mesh. The samples were coated using an osmium plasma ion coater (OPC80N; Filgen, Nagoya, Japan) and SEM observations were performed using a JSM-7500F system (JEOL, Tokyo, Japan) at 5 kV.

TEM
The male strobili in FAA solution (formalin:acetic acid:70% ethanol = 5:5:90) were sectioned into eight parts and washed with 0.1 M phosphate buffer (pH 7.4). Then, the male strobilus was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 48 h (decompression using a syringe for the first 3 h) at room temperature and then fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 h at 4°C. Following a wash with 0.1 M phosphate buffer (pH 7.4), the strobilus was further postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature and then rinsed with distilled water. The male strobilus was dehydrated using graded concentration of ethanol solutions and immersed in propylene oxide twice for 1 h each. To replace the epoxy resin (Quetol651, Nissin EM, Tokyo, Japan), the male strobilus was embedded in the resin with propylene oxide. Specifically, they were immersed for 2 h in 1/3 Quetol, 2 h in 2/3 Quetol, and 1 h in 3/3 Quetol, and then they were left to stand for 4 days while being replaced with 3/3 Quetol once a day (decompression using a syringe only on the first day) and were polymerized at 60°C for 48 h. Ultrathin sections (75 nm) were obtained using an Ultramicrotome (EM UC7; Leica Microsystems, Wetzlar, Germany) and stained with 2% uranyl acetate and 1% lead citrate.

Fine mapping of the MS4 locus
Linkage maps were constructed using a MS4 mapping family consisting of 46 normal pollen production offspring and 48 male sterile offspring produced by artificial crossing ("S8" × "S8HK5"; Table S2). The SNP marker AX-174140699 on CjTKPR1 was previously mapped on LG4 with five adjacent markers (29). These markers were developed from transcript sequences blasted against two versions (0.11 and 0.1) of draft genome assemblies of C. japonica, leading to the identification of three genomic contig sequences (ctg370 and ctg2011 from ver. 0.11 and ctg482 from ver. 0.1). These three genomic contig sequences were manually combined and genomic coordinates were calculated, starting from AX-174206035 ( Fig. 2A; Table S3). Transcript sequences from C. japonica (CJ3006NRE) (35) were mapped against genomic contig sequences using the minimap2 tool and the "-ax splice" option, resulting in 67 transcripts (Table S3). An additional marker (CJt085666 ; Table S3) was genotyped for the mapping family using PCR-restriction fragment length polymorphism (RFLP) and the NdeI restriction enzyme, to obtain CjTKPR1 and the genetic markers on either side ( Fig. 2A).

RT-PCR
cDNA was synthesized from 500-ng aliquots of total RNA from Japanese cedar and A. thaliana using the PrimeScript IV 1st Strand cDNA synthesis kit (Takara Bio, Shiga, Japan). CjTKPR1 and EF1-α were amplified using intron-containing primers to distinguish between cDNA and gDNA amplification. The primer sequences are listed in Table S5.

In situ hybridization
Full-length cDNA of CjTKPR1 was cloned into the pZErO vector (Invitrogen, Waltham, MA, USA). Subsequently, 150-bp DNA fragments were amplified from the cloned full-length cDNA using gene-specific primers containing SP6 or T7 RNA polymerase promoter sequences at the 5′-end (Table S5). PCR products were purified through phenol/chloroform/isoamyl alcohol extraction followed by ethanol precipitation and used as templates for in vitro transcription. Sense and anti-sense digoxigenin-labeled probes were synthesized using the DIG RNA Labeling Kit (SP6/ T7; Roche, Basel, Switzerland) according to the manufacturer's instructions. Male strobili of WT ("Nakakubiki-4") and ms4 mutant ("P380") plants were fixed in FAA solution (formaldehyde:acetic acid:70% ethanol = 5:5:90) at 4°C for 24 h. After dehydration and embedding, the strobili were cut to a thickness of 20 μm. Hybridization and immunological detection were performed as described previously (49,50).

Histological analysis of male strobili
Male strobili were collected from "Santo-1" (WT) and "Shindai-8" (ms4 mutant) plants, which were grown at the Niigata Prefectural Forest Research Institute. Male strobili were fixed with FAA, dehydrated using graded ethanol series, introduced to chloroform through a chloroform-ethanol series, and embedded in paraffin. We cut 10-μm sections using a microtome (ESM-100L; ERMA, Tokyo, Japan), which were then stained with Mayer's hematoxylin solution and mounted in Eukitt mounting medium (ORSAtec, Bobingen, Germany). These sections were observed under a light microscope (BX-50; Olympus) equipped with a digital camera (Wraycam-NOA2000; Wraymer).

Transgenic experiments
Total RNA of CjTKPR1 was extracted from male strobili of Ms4/ms4 heterozygous individual ("S8TM4"; Table S1). cDNA was synthesized using the PrimeScript II kit (Takara Bio). Total RNA and genomic DNA from A. thaliana (Col-0 accession) were extracted from flower buds and leaf tissues, respectively. Coding regions of CjTKPR1 and AtTKPR1 were amplified using PrimeStar GXL polymerase (Takara Bio) with specific primers (Table S5). pENTR1a, AtTKPR1 promoter (pro), coding sequences of CjTKPR1 or AtTKPR1, and Nos terminator (NosT) fragments were combined with In-Fusion HD mix (Takara Bio) and cloned to Escherichia coli. Single-nucleotide substitution constructs were made by inverse PCR with mutation-containing primers (Table S5). Each AtTKPR1pro:TKPR1 coding sequence:NosT construct was further cloned into a pFAST-G01 vector using Gateway LR clonase II (Thermo Fisher Scientific, Waltham, MA, USA). We prepared AtTKPR1/Attkpr1-1 heterozygous plants (Col-0 background; accession no. CS837358) as the transformation hosts because the Attkpr1-1 homozygous mutant produces no pollen and therefore no progenies. Each construct was transformed into AtTKPR1 heterozygous mutants via Agrobacterium tumefaciens (GV3101) using the floral dip method (51). AtTKPR1 heterozygous individuals containing an external TKPR1 construct were selected based on fluorescence in the seed and confirmed by PCR (Table S5). For the next generation, we selected Attkpr1-1 homozygous mutants with external TKPR1 sequences. Pollen production was analyzed based on anther observation under a microscope after Alexander staining (44).